Optimization Of Prokaryotic Expression Of Xinjiang Bovine Theileria Annulata Tams1 Gene And The Research Of Immunological Reactivity

Theileriosis is economically important and affects an estimated 250million cattle across an area encompassing North Africa,Southern Europe,the Indian subcontinent,Middle East,the Middle Central Asia and other place.Theileria annulata is a tick-borne protozoan parasite that multiplies in erythrocytes,causing mild hyperthermia and anemia.When infected calves are under stress,such as mixed infection with other parasites or viruses,they show severe symptoms and occasionally die.Theileriosis is one of the most economically important diseases of grazing cattle in Xinjiang area.The following two studies were conducted in the present article.1.Optimization of Prokaryotic Expression of Xinjiang Bovine Theileria annulata Tams1 GeneIn order to improve the expression level of Xinjiang Bovine Theileria annulate Tams1 gene in Escherichia coli,the effects of various expressing conditions including inducing temperature,inducing time,and the final concentration of inductor IPTG on the expression of GST-Tams1 fusion protein were studied in this paper.The results were shown the expression of GST-Tams1 fusion protein was reached to the highest level(2.5mg/mL) in bacterial strain Bl21(DE3) after pre-culture for 2.5h when 2mmol/L IPTG as final concentration was added and continuely cultured for 8h at 25℃.The SDS-PAGE analysis was shown the molecular mass of GST-Tams1 fusion protein purified by GST-Sepharose 4B affinity chromatography was about 56 KDa,conforming to the expected molecular mass of GST-Tams1 fusion protein..2.Construction of Tams1 Gene Eukaryotic Expression Vector in Xinjiang Bovine Theileria annulata and Immunological Reactivity ResearchA 846 bp Tams1 gene fragment was obtained from the whole blood genome in the infected cattle by PCR amplification and transformed into eukaryotic expression vector pcDNA3.1(+) to construct the recombineation plasmid. After sequencing and identifying,the transient expression of Tams1 was performed by injecting mice in vena caudalis with pcDNA3.1(+)-Tams1.The total RNA was extracted from murine livers.The target swap was obtained by RT-PCR from the total RNA.Then the mice were subcutaneouly injected with recombination plasmid pcDNA3.1(+)-Tams1 for four times and the antibody in mirine sere was detacted.The results were shown there was complete Tams1 gene fragments in the recombinant plasmid peDNA3.1(+)-Tams1.The specific band of antigen antibody complex was detected by Western-blot test,suggesting the recombinant plasmid pcDNA3.1(+)-Tams1 had the immunogenicity.3.The Research of Immunological Reactivity of Xinjiang Bovine Theileria annulata Tams1 fusion proteinUsing ELISA method showed that the best concentration of antigen peridiumed is 2.5μg/ml,serum dilution is 1:20. Fusion protein GST-Tams1 antiserum titer results of the assessment shows that with the increase in the number of immune or four protein immune serum liter gradually increased.The highest titer of serum is ginsenoside for adjuvant immunohistochemical,and Freund's adjuvant for the immune adjuvant group follow it;The group of injection of recombinant eukaryotic plasmid pcDNA3.1(+)-Tams1 Immunohistochemical and the group of injection of fusion protein GST-Tams1 has the similar immune serum titer.But the control group that were injected with normal saline and empty plasmid pcDNA3.1(+)is very low,almost no Antibody was produced..